The cell's ability to sense the environment and to determine the direction and proximity of an extracellular stimulus, followed by correct movement, is fundamental not only for neural development (e.g. neuronal migration and growth cone guidance) but also for immunity, angiogenesis, wound healing, and embryogenesis. Directional cell movement is also crucial for many pathological events, especially cancer-cell metastasis. Therefore, a better understanding of the cellular mechanisms that underlie the directional responses of cells to extracellular stimuli would constitute a major advance of our basic knowledge on directional cell motility and could provide the foundation for developing strategies and treatments for many illnesses. The proposed study will use nerve growth cones as the model to study the spatiotemporal Ca2+ signaling mechanisms underlying directional motility in response to extracellular cues. Calcium is a key second messenger that regulates a variety of cell motility, including directed cell migration. It has been established that Ca2+ mediates growth cone responses to guidance cues, including attractive and repulsive turning responses. Recent studies indicate that different, localized Ca2+ signals elicit a balancing act on the activity of calcium-calmodulin- dependent kinase II (CaMKII) and Calcineurin (CaN) phosphatase to control the attractive and repulsive turning of the growth cone. This application aims to further evaluate the Ca2+ mechanisms that control bidirectional growth cone steering in response to guidance cues. Three specific aims are proposed: (1) to examine the spatiotemporal patterns of cytosolic Ca2+ signals and their role in controlling growth cone steering, (2) to investigate the downstream mechanisms that sense various Ca2+ signals to control growth cone turning, (3) to test the hypothesis that FAK/Src links Ca2+ signaling to tyrosine phosphorylation in growth cone guidance. The proposed studies will take advantage of our rigorous assays of growth cone turning and a combination of high-resolution digital imaging, photoactivation of caged compounds, and molecular manipulation of signaling components. In particular, direct manipulation of intracellular Ca2+ concentrations by focal laser-induced photolysis (FLIP) of caged Ca2+ will be extensively used for dissecting the signaling components. Together, these experiments represent a comprehensive study that aims to understand the Ca2+ signaling mechanisms underlying growth cone motility and guidance. The long-term goal is to understand the molecular and cellular mechanisms that allow axonal growth cones to navigate through complex extracellular spaces for establishing intricate connections. Results from this study will not only advance our knowledge of molecular mechanisms underlying precise neuronal wiring during brain development and recovery, but also provide important insights into the cellular mechanisms underlying directional sensing of migrating cells during important biological responses such as chemotaxis of leukocytes during inflammatory response. PUBLIC HEALTH RELEVANCE: The cell's ability to sense the environment and to determine the direction and proximity of an extracellular stimulus, followed by correct movement, is fundamental not only for neural development (e.g. neuronal migration and growth cone guidance) but also for immunity, angiogenesis, wound healing, and embryogenesis. Directional cell movement is also crucial for many pathological events, especially cancer-cell metastasis. Therefore, a better understanding of the cellular mechanisms that underlie the directional responses of cells to extracellular stimuli would constitute a major advance of our basic knowledge on directional cell motility and could provide the foundation for developing strategies and treatments for many illnesses. The proposed study will use nerve growth cones as the model to study the spatiotemporal Ca2+ signaling mechanisms underlying directional motility in response to extracellular cues. The results from this set of studies will provide significant insights into the cellular mechanisms of growth cone pathfinding, as well as of directed cell movement in many physiological and pathological events. Therefore the work is directly relevant to public health.